The world is in the middle of an obesity epidemic, which appears to be followed by an epidemic of type 2 diabetes. Although lifestyle modification to induce weight loss is a cornerstone of preventing and treating diabetes, inducing a substantial and sustained weight loss has remained highly challenging. Therefore, additional drugs are required to effectively manage diabetes and its complications. However, the drug treatment of diabetes offers additional challenges. Diabetes is often associated with impaired insulin signaling, leading to a reduction in glucose uptake by adipose tissue and skeletal muscle and an increase in release of glucose in circulation, collectively contributing to the raised blood glucose levels encountered in diabetes. Moreover, diabetes is associated with increased deposition of fat in the liver, which may further impair hepatic function. Considering these challenges, a drug that can increase glucose uptake by adipose tissue and skeletal muscle, reduce hepatic glucose output and fat accumulation, even in presence of impaired insulin signaling and obesity, would be highly desirable for effective management of diabetes. Proteins of Ad36, a human adenovirus appear to offer a great template to design such a drug, as described below.
Briefly, in animal models, experimental infection with Ad36 significantly increases adiposity, but improves glycemic control and attenuates lipid accumulation in the liver, despite high fat diet (Dhurandhar N. V., et al., Obes Rev., 14(9):721-35 (2013); Krishnapuram R., et al., Am J Physiol Endocrinol Metab., 300(5):E779-89 (2011); Dhurandhar N. V., Lancet Infect Dis., 11(12):963-69 (2011); Pasarica M., et al., Obesity (Silver Spring), 14(11):1905-13 (2006)). Humans, who are naturally exposed to Ad36 infection show cross-sectional and temporal associations with obesity and better glycemic control and lower hepatic lipid levels (Krishnapuram R., et al., Am J Physiol Endocrinol Metab., 300(5):E779-89 (2011); Lin W., et al., Diabetes Care, 36(3):701-07 (2013); Yamada T., et al., PLoS ONE, 7(7):E42031 (2012); Shang Q., et al., Obesity (Silver Spring), 22(3):895-00 (2014); Trovato G. M., et al., Int J Obes. (Lond), 33(12):1402-09 (2009); Trovato G. M., et al., Liver Int., 30(2):184-90 (2010); Trovato G. M., et al., Dig Dis Sci., 57(2):535-44 (2012)). Cell culture studies indicate that the E4orf1 protein of Ad36 is necessary and sufficient for the effects of Ad36 on adiposity, glucose disposal and hepatic lipid content (Rogers P. M., et al., Int J Obes. (Lond), 32(3):397-06 (2008); Dhurandhar E. J., et al., PLoS ONE, 6(8): E23394 (2011); Dhurandhar E. J., et al., PLoS ONE, 7(10):E47813 (2012)). Working independent of the proximal insulin signaling (Krishnapuram R., et al., Int J Obes., 37(1):146-53 (2013)), Ad36 E4orf1 increases uptake of glucose by preadipocytes, adipocytes, and myoblasts, and it reduces glucose release from hepatocytes (Rogers P. M., et al., Int J Obes. (Lond), 32(3):397-06 (2008); Dhurandhar E. J., et al., PLoS ONE, 6(8):E23394 (2011); Dhurandhar E. J., et al., PLoS ONE, 7(10):E47813 (2012)). Moreover, Ad36 E4orf1 promotes lipid export, reduces lipid uptake, and promotes lipid oxidation in hepatocytes (Dhurandhar E. J., et al., PLoS ONE, 7(10):E47813 (2012)), which may collectively reduce hepatic lipid storage. Recent data show that E4orf1 can improve hyperglycemia in high fat fed mice (Ha-Na N., et al., Obesity Week, 2013, S211). Importantly, this action of E4orf1 appears to be independent of proximal insulin signaling, high fat diet, or weight loss.
As the epidemic of obesity continues unabated, infectobesity, obesity of infectious origin, has been receiving increasing attention in the recent years (Rossner S., Lakartidningen, 102(24-25):1896-8 (2005); Astrup A., et al., Int J Obes Relat Metab Disord., 22(4):375-6 (1998); Powledge T. M., Lancet Infect Dis., 4(10):599 (2004)). Although many factors contribute to the etiology of obesity, a subset of obesity may be caused by infections. In the last two decades, 10 obesity-promoting pathogens have been reported (Dhurandhar N. V., et al., Genetics and Hormones, 20(3): 33-39 (2004)). The first human virus, adenovirus type 36 (Ad-36), was reported that caused obesity in experimentally infected animals (Dhurandhar N. V., et al., Int J Obesity, 24:989-96 (2000); Dhurandhar N. V., et al., Int J Obesity, 25:990-96 (2001); Dhurandhar N. V., et al., J Nutrition, 132:3155-60 (2002)) and showed association with human obesity (Atkinson R. L., et al., Int J Obesity, 29:281-86 (2005)). In-vitro experiments have shown that Ad-36 infection of rat preadipocytes (3T3-L1) and human preadipocytes promote their proliferation and differentiation (Vangipuram S. D., et al., Obesity Research, 12:770-77 (2004)).
Ad-36 stimulates preadipocytes (pre-fat cells) to differentiate into adipocytes (fat cells), and increases the number of fat cells and their lipid content (Id.). Ad-36 can induce differentiation of preadipocytes even in absence of conventional differentiation inducers such as the cocktail of methyl isobutyl xanthine, dexamethasone, and insulin (MDI). A similar effect of the virus is observed in human adipose derived stem cells (Id.). Rats infected with Ad-36 showed greater adiposity but paradoxically lower insulin resistance 7 months post-infection (Pasarica M., et al., Obesity Research, 12 (supplement):A122 (2004)). Moreover, fat cells from uninfected rats when infected with Ad-36 show increased glucose uptake, indicating greater insulin sensitivity (Dhurandhar N. V., et al., Obesity Research, 11:A38 (2003)).
Factors required for increased insulin sensitivity include greater preadipocyte number and differentiation, and activation of cAMP and insulin signaling pathway enzymes (e.g., phosphotidyl inositol-3 kinase (PI3K or PI3 kinase)). Preadiopcyte differentiation in turn is modulated by activation of PI3 kinase and cAMP signaling pathways (Hansen J. B., et al., J Biol Chem., 276(5):3175-82 (2001); Reusch J. E., et al., Mol Cell Biol., 20(3):1008-20 (2000); Chiou G. Y., et al., J Cell Biochem., 94(3):627-34 (2005); Cornelius P., et al., J Cell Physiol., 146(2):298-08 (1991); Burgering B. M., et al., Nature, 376(6541):599-02 (1995); Magun R., et al., Endocrinology, 137(8):3590-3 (1996)). Ad-36 has been shown to increase preadipocyte replication, the number of differentiated adipocytes, and PI3 kinase pathway (Pasarica M., et al., FASEB J, 19(4):A70 (2005)).
The liver has a predominate role in fat metabolism and normally accumulates lipids (fat), but only to “normal levels.” Excessive lipid accumulation in hepatocytes results in hepatic steatosis, which is metabolically harmful and can result from a variety of liver dysfunctions, such as decreased beta-oxidation or decreased secretion of lipoproteins. Another of the many functions of the liver is to release glucose into circulation. In healthy individuals, liver cells release glucose regularly to regulate blood glucose levels. In contrast, in individuals with diabetes, liver cells release glucose uncontrollably, which increase blood glucose levels. Therefore, reducing glucose release from liver cells (hepatocytes) can be very effective in controlling diabetes.
Excessive lipid accumulation in the liver may contribute to insulin resistance, a condition in which insulin has decreased effectiveness lowering blood sugar, and thus poor glycemic control. Adiponectin, a protein secreted by fat tissue (adipose tissue) improves insulin sensitivity in many ways. Adiponectin acts via adiponectin receptors AdipoR1 and AdipoR2 in the liver to activate AMPK and PPARaC pathways (Heiker, J. T., et al., Biol. Chem., 391:1005-18 (2010)), to decrease systemic and hepatic insulin resistance, and to attenuate liver inflammation and fibrosis (Heiker et al.). It is a strong determinant of hepatic lipid content, as indicated by mice models of adiponectin “knock-out” or overexpression (Nawrocki, A. R., et al., J. Biol. Chem., 281:2654-60 (2006); Kim, J. Y., et al., J. Clin. Invest., 117:2621-37 (2007)). Adiponectin is thought to lower hepatic steatosis by the up-regulation of AMPK-mediated hepatic lipid oxidation (Xu, A., et al., J. Clin. Invest., 112:91-00 (2003)).
Non-alcoholic fatty liver disease (NAFLD) affects up to 20% of adults in the U.S., and includes the excessive accumulation of fat in the liver (hepatic steatosis). It is often associated with obesity and insulin resistance (Fabbrini, E. et al., Proc. Natl. Acad. Sci., USA 106:15430-35 (2009); Deivanayagam, S. et al., Am. J. Clin. Nutr., 88:257-62 (2008)). The prevalence of NAFLD is about 70-80% in adults with type 2 diabetes or obesity (Targher, G., et al., Diabetes Care, 30:1212-18 (2007); Bellentani, S., et al., Dig. Dis., 28:155-61 (2010); Parekh, S. et al., Gastroenterology, 132:2191-07 (2007)), 3-10%, in all children, and up to 40-70% in obese children (Bellentani et al.). NAFLD is associated with greater overall and liver-related mortality (Adams, L. A., et al., Gastroenterology, 129:113-21 (2005); Ekstedt, M., et al., Hepatology, 44:865-73 (2006)). In addition to steatosis, inflammation and fibrosis can develop and NAFLD may progress to non-alcoholic steato-hepatitis (NASH), cirrhosis, liver failure and hepatocellular carcinoma. While steatosis is potentially reversible, once it progresses to NASH, there are no established treatments, and the few available medications show limited success (Gupta A. K., et al., J Diabetes Complications, 2009; Sanyal A. J., et al., N Engl J Med., 362:1675-85 (2010)). Therefore, the timely prevention and/or treatment of hepatic steatosis is critical. However, even for NAFLD, drug treatment has marginal success (Duvnjak M., et al., J Physiol Pharmacol., 60 Suppl 7:57-66 (2009)), and reducing dietary fat intake and obesity are the mainstay of treatment (Mishra P., et al., Curr Drug Discov Technol., 4:133-140 (2007)). Despite the obvious health benefits, compliance with lifestyle changes to achieve sustained improvements in diet or obesity has proved challenging for the general population.
While excess adiposity or a high fat (HF)-diets are risk factors for NAFLD, Adenovirus 36 (Ad36) attenuates hepatic steatosis in mice despite a continued HF-diet and without a reduction in visceral or subcutaneous adiposity. Ad36 appears to qualitatively alter the metabolic quality of adipose tissue to attenuate HF-diet induced hepatic steatosis. This change in the metabolic quality of adipose tissue by Ad36 includes greater uptake and reduced release of fatty acids and greater adiponectin secretion (Rogers, P. M., et al., Diabetes, 57:2321-31 (2008); Pasarica M., et al., Stem Cells, 26:969-78 (2008)). The thiazolidinedione (TZDs) class of drugs can also improve metabolic quality of adipose tissue, up-regulate adiponectin, and improve hepatic steatosis (Nawrocki, A. R., et al., J Biol Chem., 281:2654-60 (2006); Lutchman G., et al., Clin Gastroenterol Hepatol., 4:1048-52 (2006); Shen, Z., et al., Am J Physiol Gastrointest Liver Physiol., 298:G364-74 (2010)). However, serious side effects of TZDs have been reported (Habib, Z. A., et al., J Clin Endocrinol Metab., 95:592-00 (2010); Ramos-Nino, M. E., et al., BMC Med., 5:17 (2007); Lipscombe, L. L. et al., JAMA, 298; 2634-43 (2007)).
Ad36 does not cause morbidity or unintended mortality in animals. In addition, Ad36 appears to have distinct advantages over the action of the TZDs, particularly in the presence of a HF-diet. Unlike the TZDs, Ad36 does not increase adiposity in HF-fed mice (Fernandes-Santos, C., et al., Pancreas, 38:E80-86 (2009); Fernandes-Santos, C., et al., Nutrition, 25:818-27 (2009)). In the presence of a HF-diet, TZDs can improve glycemic control, but they concurrently promote lipid storage in various organs, including the liver (Fernandes-Santos, C., et al., Pancreas, 38:E80-86 (2009); Todd, M. K., et al., Am J Physiol Endocrinol Metab., 292:E485-93 (2007); Kuda, O., et al., J Physiol Pharmacol., 60:135-140 (2009)). This and other side effects limit the clinical utility of TZDs.
Harnessing certain properties of viruses for beneficial purposes has been creatively used for several years, including the use of bactericidal properties of a bacteriophage virus (Hanlon, G. W., Int J Antimicrob Agents, 30:118-28 (2007)), the oncolytic ability of a mutant adenovirus (Bischoff, J. R., et al., Science, 274:373-76 (1996)), or the use of Herpes simplex virus and several other viruses for the treatment of cancers (Crompton, A. M., et al., Curr Cancer Drug Targets, 7:133-39 (2007)), alone, or with various synergistic drugs (Pan, Q., et al., Mol Cell Biochem., 304 (1-2):315-23 (2007); Libertini, S., et al., Endocrinology, 148(11):5186-94 (2007).
Therefore, a need exists for agents that improve glucose uptake and preferably do not increase adiposity.